Transmissible spongiform encephalopathy (TSE) is generally called prion disease, includes human Creutzfeldt-Jakob disease, BSE in cattle, scrapie in sheep and goat and the like, and is a disease diagnosed in a wide range of mammals. The common features of these diseases are that mortality after symptom onset is 100%, that antemortem diagnoses of these diseases are extremely difficult, and that these diseases are generally confirmed by pathological diagnosis at necropsy. However, it became clear that variant CJD (vCJD) is infected by transfusion, and a plurality of infection cases have been reported recently. Therefore, life-prolonging procedure and treatment for patients with TSE by antemortem diagnosis, and development of diagnosis of a specimen before symptom onset and antemortem diagnosis of TSE so as to prevent infection spread by horizontal disease transmission via blood and the like have been urgently-required.
The biggest reason that the development of this blood test is difficult is that the most appropriate marker cannot be detected in blood. In general, diagnosis of TSE is performed by detecting prion protein having an abnormal structure (PrPsc or PrPres) as a surrogate marker. This PrPres is a protein molecule distinctively detected after symptom onset. The normal PrP (PrPc) has three α-helix regions in a molecule, while PrPres is a conformational isotype having a β-sheet structure in place of the α-helix structure in this PrP molecule. For the reason, PrPres is characterized by having a resistance to digestion action by proteases as well as a remarkable stability of the molecular structure thereof.
The resistance of this protein to proteases is distinctive because this protein is resistant to proteinase K (PK) which is a strong protease and can digest almost all proteins. Only a small portion of the molecule of the abnormal PrP is digested, while almost all other proteins are digested by PK. Therefore, abnormal PrP is represented by “PrPres”. The name was made by adding “res” to the end of “PrP”.
However, this PrPres is generally confirmed only in the infected sites of animals after symptom onset (the central nerve system, or may be confirmed in the reticuloendothelial system tissue in the case of vCJD). The level of PrPres in blood is extremely small, and thus PrPres cannot be detected by conventional methods. For the above reasons, a detection method by which several to several tens of ng/mL PrPres that had not been able to be detected by conventional methods can be detected, and a method by which PrPres of an individual with the disease can be clearly discriminated from PrPres of a healthy individual, and can be detected using blood. Development of a novel substance specifically binding to PrPres or a novel method for amplifying PrPres has been studied in the world so as to satisfy the requirement.
Conventional methods which had been developed to satisfy the requirement are not sufficient in terms of detection sensitivity or specificity to constitute the blood test, and thus are not practically used. Only a method for amplifying PrPres in vitro to a detectable amount so as to detect PrPres (Peptide Misfolding Cyclic Amplification; PMCA) has been introduced as a method which may be effective to constitute the blood test (Saa, P., Castilla, J., Soto, C. 2006, Science 313:92-94). However, this PMCA method has two distinctive features that (1) it takes considerable time to amplify PrPres (the detection rate of a detection case using infected hamster blood in which PrePres had been amplified for 21 days was 60%); and that (2) the brain homogenate of a healthy animal must be optionally added as a precursor of PrPres that is necessary in amplification. Regarding the feature (2), there is a possibility that neuroblast homogenate or platelet homogenate may be used in place of the brain homogenate. However, a large amount of the neuroblast homogenate or the platelet homogenate must be cultured, or the platelet of a healthy individual must be always prepared, and thus it appears to be extremely difficult for the neuroblast or platelet homogenate to be practically used. In addition, it was reported that reaction does not occur if a plasma protein is present in a reaction mixture solution in this method (Prion 2008 symposium; Madrid). Therefore, it cannot be said that there is a possibility that the blood test can be practically carried out by this method.